Analysis of narcotics and amphetamines

ABSTRACT

APPARATUS AND METHODS FOR THE DETECTION OF NARCOTIC ANALGESICS AND AMPHETAMINES WHICH EMPLOY MACRORETICULAR HEADS OF STYRENE-DIVINYLBENZENE COPOLYMER TO ADSORB THE NARCOTIC ANALGESICS AND AMPHETAMINES FROM HUMAN URINE IN A NNONIONIC MECHANISM.

Dec. 7, 1971 J1M. FUJIMOTO ETAL 3,625,652

ANALYSIS OF NARCOTICS AND AMIHETAMINES Filed March 17, 1970 2SllOOtCi-Slluoll 1 INVENTORS JAMES M. FUJIMOTO RICHARD l. H. WANG BYWWW/W ATTORNEY United States Patent O U.S. Cl. 23-230 B 4 ClaimsABSTRACT OF THE DISCLOSURE Apparatus and methods for the detection ofnarcotic analgesics and amphetamines which employ macro-reticular headsof styre-ne-divinylbenzene copolymer to adsorb the narcotic analgesicsand amphetamines from human urine in a nonionic mechanism.

BACKGROUND OF THE INVENTION 1 Field This invention relates to the fieldof urine analysis for the' detection of narcotics, narcotic metabolitesand amphetamines.

The invention described herein was made in the course of work under agrant or award from the Department of Health, Education, and Welfare.

(2) Prior art One of the prior art methods for analyzing urine samplesfor the presence of narcotics and amphetamines is by extraction withorganic solvents, but this system is time-consuming and cumbersome inits handling of solutions so that it is only suitable for researchinvestigations instead of clinical work.

A more widely-practiced technique is the so-called Dole method whichuses ion-exchange resins to separate the narcotics from a urine sampleand is fully described in volume 198, Journal of the American MedicalAssociation 115, Detection of Narcotic Drugs, Tranquilizers,Amphetamines, and Barbiturates in Urine, Dole et al. Briefly, the Dolemethod involves soaking a piece of cation-exchange paper in a dilutedurine sample for 30 minutes or more, and extracting the components fromthe paper with three consecutive buffer-solvent systems having diflerentpH values to separate the various drugs after which aliquots of thethree solvent extracts are evaporated and the three residues are takenup with a suitable solvent for analysis by thin-layer or gas-liquidchromatography.

Although the Dole method has important advantages in comparison to theliquid extraction method, it also has several disadvantages whichinhibit its utility for the rapid analysis of a large number of samples.It generally requires the use of bottles in which the cation-exchangepaper can be immersed in the sample for the initial 30- minuteextraction period and for the butter-solvent extractions. Further a gooddeal of time is consumed in the three separate buffer-solventextractions that must be performed on the paper to furnish a completeanalysis, and additional time is needed for evaporation of the threesolvent extracts.

Most users of the Dole method usually will employ only one of thebutter-solvent extractions (primarily the one specific for morphine andsimilar drugs), but even in this practice, the Dole method requiresapproximately 90 minutes to prepare one sample for chromatographicanalysis after the initial immersion of the cation-exchange paper in thesample. A further disadvantage is the amount of space and equipmentneeded to conduct the test.

3,625,652 Patented Dec. 7, 1971 The present state of the art is suchthat there is a need for a rapid, accurate test method for narcotics andamphetamines and suitable apparatus by which it can be carried out in aneconomical fashion for analysis of a large number of samples; this needexists for observation of the therapeutic administration of narcoticsand amphetamines and particularly the detection and treatment of drugabuse.

SUMMARY OF THE INVENTION Our present invention is based upon the use ofan insoluble solid having an open-cell microporous physical structureand a nonionic chemical composition to adsorb narcotics and amphetaminesfrom urine samples. According to our method, a urine sample is passedthrough a short bed or column of the nonionic microporous solid materialon which the drugs become adsorbed, the drugs are eluted from themicroporous adsorbent and thereafter identified by any suitable means,preferably thin-layer chromatography. The apparatus, which is mostsuitably in the form of a plastic film structure, includes a firstchamber through which the sample passes and a second chamber in fluidcommunication with the first chamber which contains the solid adsorbentmaterial; after a sample has passed through the second chamber thenarcotic analgesics and amphetamines are eluted from the adsorbent solidand analyzed.

We have found small beads of nonionic styrene-divinylbenzene copolymershaving a porous macroreticular structure to the particularly effectivefor the adsorption of narcotic analgesics and amphetamines as describedabove.

Our technique provides a means to detect narcotics and amphetamines inurine that has a number of important advantages in comparison to theprior art methods discussed previously. The procedure to prepare asample for analysis requires only about fifteen minutes as compared tothe ninety minutes needed for the equivalent portion of the Dole methodwhen using only a single buffersolvent extraction. A single step elutionis provided by the present invention which removes various narcoticsfrom the urine instead of only removing a narrow band of extracts. Thepresent test is sensitive without evaporation of the extracts beforethey are analyzed. Additionally, our method detects narcotic metabolitesas well as the parent narcotic compounds withiout the need to use extrahydrolysis procedures, so that it is now possible to provide afingerprint identification of a narcotic compound. A further importantadvantage is the provision of self-contained detection unit which givesthe clinic or laboratory a pre-packaged apparatus suitable for carryingout our method in a convenient, economical and rapid manner.

One of the primary objects of this invention is to provide new methodsand apparatus for the identification of narcotics and amphetamines inhuman urine; another is to provide a narcotic and amphetamineidentification means that is sensitive but which can be performedrapidly; yet another is to provide an integrated unit which will detectnarcotics and amphetamines. A more specific object is to provide theparticular apparatus and methods hereinafter claimed.

DESCRIPTION OF THE DRAWINGS The accompanying drawings depict severalembodiments of the apparatus of this invention which are meant toillustrate, not limit, the invention inasmuch as it is anticipated thatchanges can be made in the illustrated embodiments by those skilled inthe art which will remain within the true spirit and scope of thepresent invention. In the drawings:

FIG. 1 is a plan view of a pouch-like apparatus of this invention;

FIG. 2 is a top view of the article of FIG. 1;

FIG. 3 is a plan view of another form of apparatus according to thisinvention; and

FIG. 4 is a plan view showing two thin-layer chromatograms of materialsdetected by this invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS (I) Description of method Themethod of the present invention comprises, generally, the steps of (1)passing the urine sample through a small bed of macroporous beads ofstyrene-divinylbenene copolymer resin to adsorb narcotics andamphetamines from the sample, (2) eluting the narcotics and amphetaminesfrom the adsorbent solids with a suitable polar organic solvent, andthen (3) analyzing the eluate for the presence or identification ofnarcotics and amphetamines.

The styrene-divinylbenene copolymer resin is available commercially inthe form of 20 to 50 mesh (U.S. Sieve Series) beads which have a highsurface area due to their macroreticular physical porosity; the beadsare rinsed with 5% sodium chloride and 1% sodium carbonate prior topackaging to control bacteria and mold growth during storage. The resinbeads were prepared for use by rinsing with distilled water and used inthe form of a 60 mm. bed in a 130 x 11 mm. column in the examples setout below.

The adsorption step is carried out by passing the urine sample throughthe bed or column of the adsorbent resin at a fast flow rate. The methodcan be practiced with as little as ml. of urine sample, although asample as large as 250 ml. can also be used; the larger volume sampleswill increase the sensitivity of the test results, particularly if thedrug is present at low dosage levels. After the last of the urine sampleflows through the resin bed, the bed is washed with about 10 ml. ofwater. The adsorption step removes drugs and their metabolites from theurine, together with urinary pigments.

Elution of the adsorbed drug and metabolites from the copolymer resin isaccomplished with a polar organic solvent such as methanol, ethanol oracetone. In the following examples, 10 ml. of methanol was used to elutethe adsorbed materials. It was found that the adsorbed pigments provideda useful visual indicator in that they were adsorbed and eluted in thesame fraction as the drugs and their metabolites so that collection ofthe highly colored fraction also yielded the desired narcotics andamphetamines. Using 10 ml. of solvent, the colored fraction forms aboutthe first 5 to 7 ml. of eluate; in the following examples about 3 ml. ofthe most highly colored fraction of the adsorbed materials was collectedfor analysis.

The methanol fractions collected as described above were analyzed bythin-layer chromatography according to the following procedure.Sixty-microliter spots of the methanol fractions were applied to twothin-layer chromatogram sheets (Gelman SG silica gel impregnated glassmicrofiber sheets) and the spots were dried with a hot air blower.Twenty-microliter spots of urine standards were also applied to thechromatograms. The urine standards may comprise urine containing knowndrugs with metabolites, or urine containing the pure drug; however, inthe latter instance, the finger-printing obtained from the presence ofdrug metabolites is lost, as described in detail hereinafter. The60-microliter volume was applied in 3 portions, and the -microlitervolume applied in one portion without undue spread of the spots. Onesheet was developed in solvent system A (consisting of n-butanol, aceticacid and water in proportions of :3210, v./v.) for 35 min. and the otherin solvent system B (consisting of chloroform saturated withconcentrated ammonium hydroxide) for 9 min. The developed chromatogramswere dried in an oven at 80-90 C. The two TLC sheets were sprayedmoderately with the iodoplatinate reagent for visualizing the drug andits metabolites as purple, gray,

black, or pink spots on a light pink background, The iodoplatinate wasprepared according to the directions of Cochin and Daly, vol. 18Experientia, page 294 (1962) by adding 250 ml. of a 4% solution ofpotassium iodide to 10 ml. of a 10% solution of platinum chloride anddiluting with water to 500 ml. If the spots faded, the TLC was sprayedagain with iodoplatinate reagent. Solvent system A was designedprimarily for separating polar metabolites of narcotics. System Bseparated the parent compounds; R, of about 0.1, 0.4, and 0.9 wereobtained for morphine, codeine and meperidine, respectively, andpentazocine was above codeine. Because the present method detects drugmetabolites in addition to the parent drug itself, each drug will show acharacteristic series of spots on the TLC sheets, particularly thesheets developed in solvent system A. Each dot of the chromatogramdeveloped with solvent system A comprises a metabolite, not all of whichare presently identified. Because most samples gave multiple spots, afingerprint identification is possible by direct comparison of thepattern of spots obtained with an unknown urine sample to that obtainedfrom the urine standards.

The following examples will further illustrate the present invention; ineach example, the urine samples were coded so that the person conductingthe analysis had no knowledge of the identity of the drug.

Example 1.Thirty samples of urine from patients known to be receivingmeperidine (including anileridine) at doses of 25, 50, 75 and 100 mg.,im. were rocessed according to the method described above. The drug wascorrectly identified in 27 of the samples.

Example 2.-Thirty-four samples of urine from patients receiving codeine(including oxycodone) at doses of codeine phosphate of 30 or 60 mg.,p.o., were tested. Twenty-nine samples were identified correctly.

Example 3.Three samples of urine from patients receiving morphinesulfate at a dose of 10 mg., im. were tested. The drug was correctlyidentified in two of the samples.

Example 4.-The present invention is also useful for the detection andidentification of amphetamine which, while not legally classified as anarcotic, is subject to drug abuse. Eight urine samples from patientsreceiving doses of 2.5, 5.0 and 10.0 mg. taken orally were processed bythe method described above except that the methanol eluate wasevaporated to a small volume in an oven at C. The concentrated solutionwas spotted onto the same TLC paper and developed with solvent B, afterwhich the chromatogram was sprayed with bromo cresol green (0.25 gm. inml. of methanol); the amphetamine appeared as a blue spot. All eightsamples were correctly identified as containing amphetamine. Six controlsamples analyzed at the same time also were correctly identified ascontaining no amphetamine. The sensitivity of the procedure was 1microgram of amphetamine per ml. of urine, using 50 ml. urine.

Example 5.-Thirty-nine urine samples from persons receiving pentazocineat 15, 25 and 30 mg. doses, im., were analyzed by the above method. Uponcomparison of the TLC sheets to a pentazocine standard, the drug wascorrectly identified in twenty-seven of the samples. Althoughpentazocine is not presently considered a narcotic, it can produce drugdependency of the morphine-type and may have drug abuse potential, seeSandoval and Wang, Tolerance and Dependence on Pentazocine, vol. 280 NewEngland Journal of Medicine 1391 (1969).

FIG. 4 illustrates the type of TLC fingerprinting obtained with thepresent invention. Chromatogram 39 depicts the type of spot patternresulting upon development with solvent system A and chromatogram 40showing that produced by development with solvent system B for Examples1, 2, 3 and 5. The latter solvent system is primarily for identificationof the parent compounds although it also yields some metabolites, whilesolvent system A is intended for identification of metabolites.

The letter O designates the origin and the legend S.F. indicates thesolvent front. The set of chromatogram spots referred to as pure drugswere obtained from urine containing the respective drugsin the pure orunmetabolized form.

In each of the foregoing examples, the drugs which were tested werepresent in the samples at therapeutic dose levels, which representrelatively low quantities. When a drug is being taken in an abusivemanner by a drug addict, it will be used in much greater quantitiesusually on the order of two to three times therapeutic levels. Theability of the present method to detect drugs when administered in smalltherapeutic amounts indicates it will be useful to detect and monitordrug addiction. The method is sensitive to about 0.2 microgram per ml.of urine for morphine and similar drugs, using 250 ml. of urine sample.As shown above, it is not necessary to evaporate the eluates, butevaporation can be used if so desired and the sensitivity will beincreased. It should also be noted that heroin will analyze as morphinebecause of the manner in which it is metabolized in the body.

(II) Description of apparatus FIGS. 1-3 illustrate two forms ofapparatus according to the present invention.

FIG. 1 shows a fiat funnel shaped pouch 1 formed of two superimposedsheets of flexible plastic film, such as polyvinyl chloride film, heatsealed together along edge scams 2 and 3 to form a container having afirst chamber 4 and a second chamber 5. A stiffening member 6 isattached to the top edge of the pouch 1 and includes a tab 7 having anaperture 8 for hanging the pouch. The second chamber 5 contains adesired quantity of beads 9 of styrenedivinylbenzene resin of the typedescribed above in the form of a small bed held in place by plugs 10 and11 of glass wool or similar porous material. When the pouch 1 is to beused to test a urine sample, the pouch is opened to the rounded shape asshown in FIG. 2, the stiffener 6 serving to hold the pouch in thiscondition, and the sample is poured through the first chamber 4 to passthrough the second chamber 5 for adsorption on the beads 9. After thesample has drained through completely, the adsorbent beads are eluted asdescribed previously for analysis of the narcotics. The pouch can bestored flat prior to use and also can .be folded flat after the samplehas been processed. An advantage of the present invention is that thepouch will be dry after adsorption takes place which lends itself torelatively convenient handling.

FIG. 3 illustrates a second container according to the present inventioncomprising a pouch 15 formed of two sheets of material heat sealedtogether along side seams 16 and 17 and bottom seam 18 to formrectangular container. Heat seal seams 19 and 20 join the two sheetstogether near the top of the pouch, each comprising a first oblique seamportion 21 joined to a vertical seam portion 22 which in turn isconnected to a second oblique seam portion 23. The pouch is therebydivided into a first chamber 25 which has a generally funnel-shape, asecond smaller chamber 26 and a relatively large third chamber 27. Aquantity of beads 9 is contained in the second chamber 26 and held inplace by plugs 31 and 32 glass wool or similar porous or fibrousmaterial. The top of the pouch 15 may be stiffened in the manner shownin FIGS. 1 and 2. After the sample passes through the first chamber 25and is adsorbed on the resin contained in the second chamber, it iscollected in the third chamber 27. The chamber 2-7 can include an outletat the bottom for discharge of the sample; otherwise, the pouch 15 canbe severed near the bottom of the second chamber 26' and the uppersection preserved for analysis while the lower section is emptied ordiscarded; perforations can be formed partly across the pouch just abovethe third chamber to aid in its severance from the rest of the pouch.

Plastic films such as polyvinyl chloride, nylon, polyethylene,polypropylene, etc., which can be sterilized are suitable for thepouches 1 and 15. The film may be inherently heat scalable or caninclude a functional heat seal coating; laminated, coated or monofilmsheet material may be used. The size of the several chambers will dependupon the quantity of sample likely to be processed and the quantity ofadsorbent solids used in the second chamber. It is also desirable thatthe film material be capable of being written on so that each pouch canbe suitably identified.

There has thus been described methods and apparatus for the detectionand identification of narcotic analgesics and their metabolites andamphetamines in urine based upon the adsorption of such compounds on aspecified class of solid adsorbents. The adsorbent for the practice ofthe present invention is to comprise a solid material having a nonionicchemical composition and a macroreticular physical structure having ahigh surface area. The adsorbents are in the form of beads, each beadcomprising an agglomeration of a large number of very small microspheresto provide an open-cell porous structure. The adsorbed materials arecollected generally within the surface areas of the adsorbent and can bereadily eluted therefrom with suitable solvents. A specific class ofcompounds of this definition consists of macroreticular beads ofstyrene-divinylbenzene copolymer resins. These are commerciallyavailable, for example, from Rohm and Haas Company under the trade namesAmberlite XAD1, XAD-2 and XAD-4. These resins have an average porediameter ranging from 50 to 205 angstrom units and a surface area offrom to 850 square meters/gram. The XAD-2 styrene-divinylbenzenecopolymer resin used in the preceding examples had a surface area of 315square meters/ gram, an average pore diameter of 91 angstrom units, anda porosity of 0.43 ml. pore/ml. head. The drug compounds are held by theadsorbent primarily by Van der Waals type forces. Adsorbent resins ofthis composition are also capable of adsorbing pigments from the urinesample so that it is possible for the technician performing the test tohave a visual indication of the most suitable fraction to be eluted foranalysis.

The present technique may be utilized to analyze for narcotic analgesicsin general, particularly as defined under the Harrison Narcotic Act.These are largely morphine-like drugs including morphine derivatives andmorphine surrogates. This will thereby encompass drugs and narcoticsreceiving wide therapeutic use and will also include drugs which aresubject to drug abuse or addictive use. Amphetamines, includingamphetamine and methamphetamine, also can be detected by the presentmethod which is particularly important because of their widespreadabusive use. Detection of drug metabolites with the present inventionrequires no extra steps such as hydrolysis or other techniques, and theconcurrent adsorption of metabolites enables the use of a convenientfingerprinting system for identification of the drugs, thereby enhancingthe accuracy of the final result.

The narcotic and amphetamine analysis set forth herein represents a testmethod which can be easily performed and is particularly useful whenlarge numbers of urine samples are to be analyzed because it can beperformed rapidly. For example, it is presently believed that a trainedtechnician can analyze fifty urine samples in three hours with thepresent invention. Furthermore, the types of apparatus described hereinprovide self- 'contained test units which are convenient to store anduse.

We claim:

1. As an article of manufacture, a container formed of plastic sheetmaterial joined together to define a unit suitable for the analysis ofnarcotic analgesics and amphetamines in a urine sample comprising, incombination:

a first chamber having an open top through which the urine sample can beintroduced, and a second cham;

ber connected to the first chamber,

a bed of a solid adsorbent comprising beads of styrenedivinylbenzenecopolymer in a second chamber, said copolymer having a nonionic chemicalcomposition and a macroreticular physical structure to adsorb narcoticanalgesics and their metabolites and amphetamines from a urine samplewhich passes through (1) adsorbing the narcotic analgesics, theirmetabolites and amphetamines from a urine sample by passing the urinesample through a bed of solid beads of styrene-divinylbenzene copolymerhaving a chemically nonionic composition and a macroreticular physicalstructure, and

the second chamber. 2. As an article of manufacture, a container formedof plastic sheet material joined together to define a unit vent foranalysis of the eluate. suitable for the analysis of narcotic analgesicsand am- 10 4, A method according to claim 3, including the stepsphetamines in a urine sample, comprising, in combination: f;

a first Chambfif having an p p through Which the adsorbing pigments froma urine sample along with i116 Sample can be introduced, and a SecondChamthe narcotics, metabolites and amphetamines, and her connected tothe first chamber, eluting the colored fraction of the adsorbedmaterials a bed of solid adsorbent comprising beads of styrene- 15 f 1 idivinylbenzene copolymer in the second chamber, said copolymer having anonionic chemical composition and a macroreticular physical structure toadsorb narcotic analgesics and their metabolites and amphetamines from aurine sample which passes through 20 the second chamber, and

a third chamber connected to the second chamber to receive a urinesample after it has passed through the second chamber.

3. A method for the detection of narcotic analgesics 25 and theirmetabolites and amphetamines contained in urine comprising the steps of:

(2) eluting the narcotic analgesics, metabolites and amphetamines fromthe adsorbent solid with a sol- References Cited E. E. Hamlow et al., J.Am. Pharm. Assn., XLIII, No. 8, 460-4 (1954).

Analytical Abstracts, vol. 15, Abs. 3471 (1968).

MORRIS O. WOLK, Primary Examiner S. MARANTZ, Assistant Examiner US. Cl.X.R.

